We propose to use a new experimental model of aflatoxin hepatocarcinogenesis in glutathione S- transferase A3 knockout mice (mGSTA3-/- or KO) to explore the role of oval cells (OCs) as precursors to hepatocellular carcinoma (HCC) not possible in other experimental models. HCC is a leading cause of cancer mortality worldwide, and dietary aflatoxin B1 (AFB1) exposure is a major risk factor. Adult mice are resistant to AFB1-induced liver injury because of high levels of constitutively expressed mGSTA3 that detoxifies activated AFB1. Humans do not have this efficient detoxification pathway, so that the mGSTA3-/- mice may be considered humanized in regard to AFB1 metabolism and sensitivity. Both male and female mGSTA3 KO mice respond to low doses of AFB1 with liver injury and massive OC proliferation. In Specific Aim 1, we will define the effects of loss of the mGSTA3 on AFB1-induced toxicity, by measuring the steps of AFB1 metabolism, the levels of AFB1-DNA adducts and extent of oxidative damage in liver tissues. This will provide a mechanistic insight into AFB1 carcinogenesis and should explain the differences in susceptibility to acute AFB1 toxicity between male and female mGSTA3-/- mice that we have observed. In Specific Aim 2, four regimens of AFB1 exposure in mGSTA3-/- mice will be used to determine one, or several, that would best serve to study the AFB1-induced injury and development of HCCs. The models will be designed to target oval cells, mimic initiation + promotion protocols and include cross-breeding with chronic hepatitis-prone transgenic mice. In Specific Aim 3, the fate of oval cells after proliferation will be specifically addressed. Following AFB1- induced liver injury, their origin, expansion and differentiation into either mature cells or to liver cancer, will be studied under different AFB1 exposure protocols. The oval cell response in mGSTA3-/- mice is much greater, different in growth pattern and more relevant to human health than other mouse models reported. These studies will provide new insights into aflatoxin B1-associated carcinogenesis, identify the sequence of events that leads to tumor development, determine the role of oval cells as cancer stem cells and provide mechanisms that can be targeted to prevent AFB1-induced liver toxicity and cancer in the future.